Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways.

Acetylcholine has been well known as one of the most exemplary neurotransmitters. In humans, this versatile molecule and its synthesizing enzyme, choline acetyltransferase, have been found in various non-neural tissues such as the epithelium, endothelium, mesothelium muscle, blood cells and immune cells. The non-neuronal acetylcholine is accompanied by the expression of acetylcholinesterase and nicotinic/muscarinic acetylcholine receptors. Increasing evidence of the non-neuronal acetylcholine system found throughout the last few years has indicated this neurotransmitter as one of the major cellular signaling molecules (associated e.g. with kinases and transcription factors activity). This system is responsible for maintenance and optimization of the cellular function, such as proliferation, differentiation, adhesion, migration, intercellular contact and apoptosis. Additionally, it controls proper activity of immune cells and affects differentiation, antigen presentation or cytokine production (both pro- and anti-inflammatory). The present article reviews recent findings about the non-neuronal cholinergic system in the field of immune system and intracellular signaling pathways.

Introduction

Acetylcholine (ACh) was the first characterized neurotransmitter [1]. It is an organic molecule and one of many neurotransmitters in the autonomic nervous system (ANS). There are two main classes of acetylcholine receptors (AChR): nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors (mAChR). The nAChR is the best-characterized ligand-dependent, cation-selective channel receptor. Nicotinic acetylcholine receptors are present in the autonomic ganglia and at the neuromuscular junction. In the brain, nAChRs play an important role in the brain activity modulating synaptic and cellular functions [2], while in muscles they are involved in neural activation of muscle spasms [3]. In vertebrate species, 17 different subunits of nAChR have been identified (α1-10, β1-4, δ, ɛ, γ) [4]. The subunits form a large number of homo-and heteropentameric receptors and all these combinations show specific pharmacological properties. Stimulation of nAChRs opens channels, induces an inflow of sodium ions and/or calcium ions as well as a potassium ions outflow (Fig. 1). As a result of membrane depolarization, voltage-operated calcium channels are opened, leading to an additional flow of calcium ions. The influx of calcium causes secretion of mitogenic factors and activates cell signaling cascades [5].

Fig. 1

Schematic representation of the two most common subtypes of nAChRs. Both receptors consist of five subunits. The α7 nicotinic acetylcholine receptor (nAChR) consists of five α7 subunits and is called α7 homo-oligomeric nAChR (left). The α4β2 nAChR is composed of two α4 subunits and three β2 subunits and thus is called α4β2 heteromeric nAChR (right). In both nAChRs, subunits are arranged around a central pore or channel that opens when agents such as acetylcholine or nicotine bind to an nAChR, allowing positively charged ions to flow through the channel into the cell. The α7 nAChR principally allows passage of calcium (Ca2+) ions, whereas the α4β2 nAChR allows passage of both calcium and sodium (Na+) [16]

The terms ‘non-neuronal cholinergic system’ and ‘non-neuronal acetylcholine’ were first introduced in the 1970s by Sastry and Sadavongvivad [6]. Since that time, multiple studies have been performed to investigate the function of nACh receptors in different non-neuronal tissues. Nicotinic acetylcholine receptors were found on keratinocytes [7], blood cells including lymphocytes, macrophages, mast cells, dendritic cells, basophils [8], microglia, endothelial cells [9], epithelial cells and fibroblasts [10, 11]. The expression of this receptor was also observed in tumor cells, suggesting its potential role in cancer progression [12]. Modulation of nAChRs activity in cells from ‘non-neuronal cholinergic system’ was associated with changes in proliferation, differentiation, migration, adhesion, cell contact, apoptosis, and angiogenesis process [7, 13, 14]. The expression of nAChRs was also detected in spermatogonia, spermatocytes, seminiferous tubular and Sertoli cells. This suggests that the presence of the non-neuronal cholinergic system in testicular parenchyma plays a potentially important role in the differentiation of germ cells [15].

The role of the non-neuronal cholinergic system in the immune system

Studies performed in the past years have indicated that cholinergic transmission may modulate various aspects of both innate and adaptive immune response. It has been shown that acetylcholine – an element of the non-neuronal cholinergic system – exhibits an anti-inflammatory action. Acetylcholine may affect the immune system in two different ways – autocrine/paracrine (immune cells have the capacity of acetylcholine secretion) or nerve stimulation [17, 18].

‘Cholinergic anti-inflammatory pathway’ response plays a role in the immune response and inflammatory cascade. The non-neuronal cholinergic system affects also immune cell proliferation, T helper differentiation, antigen presentation and cytokine production. The α-7 nicotinic acetylcholine receptor (α7-nAChR), a part of the non-neuronal cholinergic system, plays a specific role in the immune response. It is localized in various immune cells, i.e. macrophages, dendritic cells, T and B lymphocytes, mast cells, or basophils [14, 19–21].

The cytokine suppressing mechanism of the inflammatory reflex – “cholinergic anti-inflammatory pathway” – requires the α-7 subunit of nicotinic acetylcholine receptor (α7-nAChR). As already known, stimulation of the vagal nerve protects from excessive cytokine production. The release of acetylcholine in spleen suppresses production of pro-inflammatory cytokines (e.g. TNF-α) and attenuates inflammation [22]. Signaling through the efferent arc of the inflammatory reflex regulates the innate immune response [23]. Interestingly, Olofsson et al. [24] have demonstrated that deficiency of α7-nAChRs in the bone marrow cells significantly impaired nerve regulation of TNF release, but α7-nAChR deficiency in neurons, or other cells had no significant effect. Authors suggested that the expression of α7-nAChR on most of the immune cells, but not in the neurons or T cells, is necessary to sustain the inflammatory reflex function. Furthermore, deficiency or impairment of α7-nAChR signaling leads to overproduction of cytokines, and enhances tissue damage [25]. Positive agonists of α7-nAChR were used in preventing tissue damage caused by the inflammatory process. Sun et al. [26] have shown that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors, reduces brain injury and improves the neurological function after focal cerebral ischemia in rats. Additionally, PNU-120596 administration had anti-nociceptive effects in mice models of persistent pain [27].

The α-7 nicotinic acetylcholine receptor is necessary for regulation of macrophage cytokine release. Macrophages isolated from the wild type mouse produced significantly lower levels of tumor necrosis factor α (TNF-α) after LPS stimulation if 5-10 minutes before they were pre-incubated with nicotine (1, 10 µM) or acetylcholine (10 µM) than macrophages isolated from α7-nAChR knockout mice [28]. In LPS-stimulated human macrophage cultures, acetylcholine attenuated release of cytokines: (TNF-α), IL-1β, IL-6 and IL-18, but not the anti-inflammatory cytokine IL-10 [29]. Similarly, Rosas-Ballina et al. [30] have shown that an α7-nAchR agonist (GTS-21) suppresses pro-inflammatory cytokine production (mainly TNF-α and IL-1β) in response to various toll-like receptor (TLR) ligands e.g. TLR2, TLR3, and TLR9 agonists. This relationship was also observed in culture of microglia cells where acetylcholine and nicotine pre-treatment inhibited lipopolysaccharide (LPS)-induced TNF-α release [31].

T cells possess all cholinergic elements of the non-neuronal cholinergic system: acetylcholine, choline acetyltransferase (ChAT), muscarinic and nicotinic acetylcholine receptors and acetylcholinesterase. B cells possess muscarinic and nicotinic acetylcholine receptors and acetylcholinesterase. ACh or nicotine treatment of T and B cells causes rapid and transient intercellular calcium ion signaling in these cells. Rosas-Bollina et al. [23] demonstrated the role of the vagus nerve and ACh in controlling the innate immune response (reduction of inflammation). The vagus nerve stimulated acetylcholine-producing memory phenotype T cells, which were required for inhibition of cytokine production. Permanent nicotine stimulation down-regulates the nAChR expression and suppresses T cell activity [32-34]. Moreover, Fujii et al. [35] showed that stimulation of the T cell receptor (TCR) using a T cell activator – phytohemagglutinin (PHA), induces ACh synthesis and release in MOLT-3 and HSB-2 cells (human leukemic T cell lines). It is well known that antagonists of the α7-nAChR, such as α-bungarotoxin (α-BTX) and methyllycaconitine (MLA), enhance T cell proliferation. Recent data indicate that α7-nAChR seems to be a key regulator of immunosuppressive functions of CD4+ CD25+ regulatory T cells [36]. Additionally, Nordman et al. [37] revealed that not only α7-nAChRs but also α4-nAChR nicotinic receptors are involved in CD4+ T-cell proliferation and helper T-cell immune response.

Activation of α7-nAChRs affect dendritic and mast cells metabolism. Studies performed in vitro in human dendritic cells after nicotine administration showed a decrease in their phagocytic activity and secretion of pro-inflammatory cytokines [38]. Nicotine inhibits IL-12 release by LPS-matured human competent dendritic cells and reduces secretion of IFN-γ by the primed T cells [39]. Simultaneously, nicotine administered to mice stimulated with prophylactic and therapeutic vaccines, adversely affects dendritic cells and prevents development and maintaining of Ag-specific effector memory Th1 cells and Ab production [40]. Studies performed on rats revealed that acetylcholine, choline and nicotine induce mast cell degranulation [41]. Mishra et al. [42] have demonstrated that nicotine affects the late, but not the early, degranulation phase of the mast cell activation. Authors suggested that nAChRs localized on mast cells might be an important component for new therapeutic approaches to control allergic diseases and allergic asthma. The α7-nAChR specific agonist GTS-21 also shows anti-inflammatory effects on mucosal-type murine bone marrow-derived mast cells (mBMMCs) and inhibits IgE-induced degranulation [43].

Nicotinic acetylcholine receptor also regulates the expression of cell surface proteins associated with regulation of leukocyte trafficking to peripheral inflammatory sites. CD11b on the surface of neutrophils is necessary for β2-integrin regulated neutrophil adhesion to the endothelium and neutrophil transmigration to sites of microbial infection [9]. It has been shown that nicotine administration and vagus nerve stimulation (VNS) significantly reduced both LPS- and fMLP-(N-formyl-methionyl-leucyl-phenylalanine) induced neutrophil surface CD11b expression via suppression of F-actin polymerization [44].

The ACh esterase plays an important role in the acetylcholine stimulation of non-neural immune cells. The enzyme is a member of the α/β hydrolase family and it degrades the excitatory neurotransmitter acetylcholine (ACh). Degradation of ACh by acetylcholinesterase (AChE) is necessary for termination of the signal and may be controlled at different cell levels. AChE can be incorrectly translocated from the cytoplasm to the membrane, which was observed in an autoimmune disease – myasthenia gravis [45]. Acetylcholinesterase function can be also modulated by an increase or decrease in its activity. Gilboa-Geffen et al. [46] in non-obese diabetic mice showed that TLR9 activated by oligonucleotide BL-7040 induced an alternative NF-κB activation mode resulting in a reduced peripheral AChE activity. Similarly, Ofek et al. [47] observed in young volunteers an increase or decrease in the plasma AChE activity 1.5 h after Escherichia coli endotoxin administration compared to saline. Moreover, AChE function can be regulated at the expression level by alternative splicing [48], microRNA suppression [49] or transcriptional control [50].

α7-nAChR and signaling pathways

The α7-nAChR subtype is activated quickly and its desensitization is fast. This receptor exhibits a higher permeability for calcium ions in relation to other nAChRs [51]. As a result of increased intracellular calcium concentrations, the cellular signaling pathways are activated leading to the inflammatory response or cancer progression [52]. The influx of calcium ions activates phosphorylation cascade via protein kinase C (PKC), serine-threonine kinase Raf-1, mitogen-activated protein kinase (MAPK) [53] and/or extracellular signal-regulated kinase ERK1, ERK2 [54]. It has been shown that many intracellular proteins interact with α7-nAChRs and participate in signal transduction [55]. As a result of nAChRs activation, nuclear transcription factor kappaB (NF-κB) is activated via the Janus kinase 2 (JAK-2) [56], phosphatidylinositol kinases PI3K and serine-threonine protein kinase AKT [57]. Other transcription factors were also activated by stimulation of nAChR: activator protein-1 (AP-1), signal transducer and activator or transcription 1 (STAT1), STAT3 and STAT5 in the JAK-2/PI3K/AKT pathway [52, 59]. The α7-nACh receptor is also engaged in Ras-Raf-ERK signaling pathway [60]. Activated nAChRs mediate carcinogenic processes including proliferation, inhibition of apoptosis, angiogenesis, migration and cell adhesion [61]. Antagonists of nAChRs may help to inhibit tumor cell proliferation [62]. Other researchers suggest that molecular therapy strategies aimed at nAChRs inhibition or siRNA silencing in lung cancer may reduce tumor growth and development [12].

Nicotine activation of the α7-nicotinic acetylcholine receptor resulted in activation of α7-nAChR–JAK2–(NF-κB; STAT3)–Bcl-2 signaling pathway and caused a reduction in the apoptosis process [56]. De Jonge et al. [58] revealed that acetylcholine released by the efferent vagal nerve inhibits macrophage activation. Stimulation of α7-nAChRs in mouse macrophages in vitro and in vivo leads to an anti-inflammatory effect via the JAK2–STAT3 signaling pathway. In turn, Wang et al. [62] have observed that activation of α7 nAChRs on human macrophages inhibits activation of the NF-κB signaling pathway induced by either endotoxin or TNF-α. This caused inhibition of pro-inflammatory cytokines release. Activation of α7-nAChR, an endogenous inflammation-resolving pathway, prevents TNF-α induced NF-κB activation and inhibits macrophage migration into the hippocampus [63]. A study by Waldburger et al. [64] shows that activation of α7-nAChRs by an agonist inhibits cytokine production by resident fibroblast-like synoviocytes (FLS), which are potentially directly involved in propagation of inflammation. In another study, Al-Wadei et al. [65] showed that nicotinic agonists – acetylcholine, nicotine, and its nitrosated carcinogenic derivative NNK activated phosphorylation of CREB (cAMP response element-binding protein), ERK, Src (proto-oncogene tyrosineprotein kinase) and AKT. Also, nAChRs stimulate growth and migration of pancreatic cancer cells.

Stimulation of α7-nAChRs by nicotine increases the expression of β-defensins in keratinocytes [66]. Li et al. [67] have shown inhibition of TNF-α expression in response to LPS after an α7-nAChR nicotine stimulation. Moreover, nicotine administration decreased the expression of primary response gene 88 protein (MyD88), NF-κB p65 protein, phospho-I-κBα, TNF-α as well as NF-κB activity induced by LPS in airway epithelial cells (HBE16). The addition of an α7-nAChR inhibitor – α-bungarotoxin caused the opposite effect. Nicotine reduced also TNF-expression through the α7 nAChR/MyD88/NF-κB pathway. In addition, Li et al. [67] revealed that when silencing of α1, α5 or α7-nAChRs in airway epithelial cells (HBE16) with appropriate siRNA, only the α7-nAChR is responsible for lowering TNF-α production.

In normal human bronchial epithelial (HBE) cells, nicotine administration resulted in phosphorylation of the signaling protein p38MAPK [68], Akt [69] and an increase in proliferation of small cell lung cancer cell lines [70]. It is well known that Akt participates in cellular processes such as, glucose metabolism, cell cycle progression, and apoptosis [71]. Nicotine has been reported to inactivate the pro-apoptotic protein Bax [72], also by the Akt signaling pathway. In HEK 273T cells, the influx of calcium stimulated by nicotine can be blocked by α-BTX (an antagonist of α7-nAChR). Activation of Akt increased phosphorylation of downstream substrates including GSK-3, p70S6K, 4EBP-1, and FKHR and could contribute to tumorigenesis in vivo in lymphoid, breast, ovarian, prostate, and brain tissues [73]. Other study showed that in neuronal cells, α7-nAChR activates the PI3K/Akt pathway by activation of a Src family member [74].

Nicotine at a concentration of 10 mM caused phosphorylation of mitogen-activated protein kinase p 44/42 MAPK in non-small cell lung cancer (NSCLC) cell lines. p44/42 MAPK is often up-regulated in cancer and can be inhibited using nAChR antagonists. Moreover, nicotine can activate α7-nAChRs to inhibit the expression of inducible nitric oxide synthase and nitric oxide through the mitogen-activated protein kinase (MAPK)/NF-κB pathway [75]. Nicotine activation of nAChR, NF-κB p65 and p50, I-κBα activation, and the MAPK signaling pathway induced vascular endothelial cells to produce adhesion molecule: ICAM-1 and VCAM-1 [76].

Cholinergic receptors stimulation via nAChR agonists, such as nicotine and GTS-21 or the vagus nerve stimulation suppresses leukocyte recruitment and the endothelial cell activation. Chatterjee et al. have shown significantly reduced production of IL-6-mediated monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 expression regulated through the JAK2/STAT3 pathway in macrovascular human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (MVECs). Authors found that cells treated with cholinergic agonists (e.g., nicotine and GTS-21) significantly reduced phosphorylation of JAK2, reduced STAT3 activation (by phosphorylation and DNA binding) and decreased levels of SOCS3 (suppressor of cytokine signaling). These changes resulted in suppression of IL-6-mediated endothelial cells activation via the JAK2/STAT3 pathway [77]. Similarly, Saeed et al. demonstrated that cholinergic agonists (nicotine) suppressed endothelial cell activation (in in vitro and in vivo studies) and leukocyte migration in a carrageenan air pouch model [78]. On the other hand, studies performed by de Jonge et al. [53] showed that nicotine induced STAT3 activation in macrophages following LPS treatment. Also Chen et al. [79] have observed simultaneous activation of Stat3 and extracellular signal regulated kinase 1/2 (ERK1/2) in T24 cells after nicotine administration. However, long-term nicotine exposure induced activation of Stat3 and down-regulation of ERK1/2 via nAChR and β-adrenoceptors in human bladder cancer cells (T24 cell line) [80].

Activation of calcium ions transport by α7-nAChRs plays an important role in the cell survival. Increase in intracellular calcium ions can cause indirect activation of β-adrenergic receptor signaling, which induces activation of epidermal growth factor (EGF) and activation of epidermal growth factor receptor (EGFR). EGFR activates the Akt pathway and its downstream effectors, X-linked inhibitor of apoptosis protein (XIAP)-survivin and nuclear factor-κB (NF-κB). Furthermore, nicotinic stimulation of α7-nAChRs confirmed the anti-apoptotic activity of these receptors by activation of PKC, PKA and NF-κB, and down-regulation of the tumor suppressor p53 [81]. NF-κB phosphorylation caused by nicotine also promotes phosphorylation of the apoptotic protein Bad and Bax and involvement of MEK and PI3K pathways [82].

Summary

Acetylcholine is produced by practically all types of the living cells. The neurotransmitter participates in numerous interrelated biologic processes such as proliferation, differentiation, apoptosis, adhesion, and migration. Both acetylcholine and nicotine (agonists of nAChR) may affect different kinds of cells by autocrine or paracrine stimulation. The cholinergic anti-inflammatory pathway is an important mechanism that inhibits cytokine production and minimizes tissue injury during inflammation. As known now, the stimulation of the vagus nerve, which releases acetylcholine, and administration of cholinergic agonists, such as nicotine and GTS-21, reduce cytokine production in the models of acute, systemic inflammation, including endotoxemia, hemorrhagic shock, ischemia- reperfusion injury, and polymicrobial sepsis. Proper inhibition or stimulation of this nicotinic acetylcholine receptor (or its selected subtype) may give comprehensive therapeutic possibilities to effective treatment of neurodegenerative as well as inflammatory, autoimmune or cancer diseases. However, for proper application of such therapies nAChR modulation in different kinds of cells should be fully understood.